Folded proteins are composed mostly of 2-sheets, 1-helices, and reverse turn conformations. Protein folding cannot be understood without knowing the structural basis for the energetics of these conformations. The long- term goal of these studies is to provide such an understanding for 2-sheet conformations. While substantial progress toward this goal has been made over the lifetime of this project, important questions remain, including the influence of N-linked glycosylation on 2-sheet folding energetics. N-glycosylation is a co-translational modification made on about 1/3 of the eukaryotic proteome before folding commences. Though we recognize the importance of the vast biology of N-glycosylation, we focus in this proposal on the following central hypotheses: (1) that the effects of N-glycosylation on 2-sheet folding energetics stem largely from specific protein-glycan interactions, and that a stabilizing structural motif containing such favorable interactions, called an enhanced aromatic sequon, can be engineered into a substantial fraction of reverse turns in proteins; and (2) that the oligosaccharyltransferase enzyme complex and the protein folding and trafficking machinery of the endoplasmic reticulum prefer to glycosylate, fold and secrete proteins with enhanced aromatic sequons, which stabilize the native state. In Specific Aim 1, we discern whether the tripartite native-state-stabilizing interaction between an aromatic side chain, GlcNAc1 of the glycan, and a Thr side chain can be applied in multiple reverse turn types. Moreover, we will alter the electronic properties of the aromatic ring, the amphiphilicity of the N-linked carbohydrate, and the hydrophobicity and hydrogen bonding potential of the Thr residue to understand the stabilizing aspects of this tripartite interaction in distinct turn types. The basis for the higher cellular N-glycosylation yields for proteins containing enhanced aromatic sequons is explored in Specific Aim 2, utilizing pulse-chase experiments to discern whether glycosylation and/or trafficking is faster and whether endoplasmic reticulum-associated degradation is slower for such proteins. We also introduce a high throughput green-fluorescence protein-lectin quenching assay as an approach to identify the sequences from a relatively large pool that afford the highest glycosylation yields in the context of reverse turns. This information is critical to render glycosylation in cell lines efficient and more homogeneous in order to make glycosylation more predictable for non-experts. The proposed work is significant because it provides engineering guidelines for adding glycans to proteins to stabilize them, to optimize their production, to extend their shelf lives, and, in the case of protein drugs, to lengthen serum half-lives, enhance protease resistance, and reduce aggregation propensity. The innovation in the proposed work is that the cell-based and in vitro assays to follow up on our observation of increased glycoprotein yield has the potential to transform our understanding of sequon usage to a more reliable guideline-based approach wherein sequon glycosylation efficiency can be predicted, enabling efficient glycoprotein production in cells and enabling non-experts to study glycoproteins. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to the public health mission because there has been a marked increase in the use of protein therapeutics over the last decade. The pharmacologic properties of many of these proteins are improved when they are N- glycosylated; however, deciding where to put the N-glycans without compromising stability or function has been largely a trial and error process with few engineering guidelines. Our discovery of the enhanced aromatic sequon, which will be significantly elaborated and improved by the proposed research plan, provides a blueprint for optimizing protein therapeutics to be more stable, less aggregation prone, and more efficiently glycosylated, folded and trafficked by the cellular machinery than their non- glycosylated analogs.